In chromaffin cells, Ca 2+ binding to synaptotagmin-1 and -7 triggers exocytosis by promoting fusion pore opening and fusion pore expansion. Synaptotagmins contain two C2 domains that both bind Ca 2+ and contribute to exocytosis; however, it remains unknown whether the C2 domains act similarly or differentially to promote opening and expansion of fusion pores. Here, we use patch amperometry measurements in WT and synaptotagmin-7-mutant chromaffin cells to analyze the role of Ca 2+ binding to the two synaptotagmin-7 C2 domains in exocytosis. We show that, surprisingly, Ca 2+ binding to the C2A domain suffices to trigger fusion pore opening but that the resulting fusion pores are unstable and collapse, causing a dramatic increase in kiss-and-run fusion events. Thus, synaptotagmin-7 controls fusion pore dynamics during exocytosis via a push-and-pull mechanism in which Ca 2+ binding to both C2 domains promotes fusion pore opening, but the C2B domain is selectively essential for continuous expansion of an otherwise unstable fusion pore. patch amperometry | synaptic transmission | capacitance measurements | neurotransmitter release | patch clamp

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Membrane fusion and fission are fundamental processes in living organisms. Membrane fusion occurs through the formation of a fusion pore, which is the structure that connects two lipid membranes during their fusion. Fusion pores can form spontaneously, but cells endow themselves with a set of proteins that make the process of fusion faster and regulatable. The fusion pore starts with a narrow diameter and dilates relatively slowly; it may fluctuate in size or can even close completely, producing a transient vesicle fusion (kiss-and-run), or can finally expand abruptly to release all vesicle contents. A set of proteins control the formation, dilation, and eventual closure of the fusion pore and, therefore, the velocity at which the contents of secretory vesicles are released to the extracellular medium. Thus, the regulation of fusion pore expansion or closure is key to regulate the release of neurotransmitters and hormones. Here, we review the phases of the fusion pore and discuss the implications in the modes of exocytosis.

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